Abstract
Cis-acting RNA structures in the genomes of RNA viruses play critical roles in viral infection, yet their importance in the bipartite genomes of the picorna-like, plant-infecting comoviruses has not been carefully investigated. We previously characterized SLC, a stem-loop structure in the 5′ untranslated region (UTR) of the bean pod mottle comovirus (BPMV) RNA2, and found it to be essential for RNA2 accumulation in infected cells. Here we report the identification of SL1, a similar cis-acting element in the other BPMV genome segment - RNA1. SL1 encompasses a portion of RNA1 5′ UTR but extends into the coding sequence for nine nucleotides, thus was missed in the previous study. While the stems of SL1 and SLC share little sequence similarity, their end loops are of the same size and identical for 11 of 15 nucleotides. Importantly, SL1 and SLC are functionally interchangeable, and separate exchanges of the stem and loop portions were likewise well tolerated. By contrast, the conserved loop sequence tolerated minimal perturbations. Finally, stem-loop structures with similar configurations were identified in two other comoviruses. Therefore, SL1 and SLC are likely essential comoviral RNA structures that play a conserved function in viral infection cycles.
Highlights
The genomes of many single-stranded, positive sense (+) RNA viruses fold into numerous secondary structures that play critical cis-acting roles in the infection cycles of these viruses[1]
We previously reported the characterization of SLC, a stem-loop structure within 5′ untranslated region (UTR) of Bean pod mottle virus (BPMV) RNA2 required for accumulation of RNA2 in infected cells[13] (Fig. 1b)
RNA1 5′ UTR, we initially speculated that SLC could be a cis-acting RNA element unique to RNA2 that facilitates RNA2 entry into virus replication complexes (VRCs) assembled by RNA1-encoded proteins[13]
Summary
The genomes of many single-stranded (ss), positive sense (+) RNA viruses fold into numerous secondary structures that play critical cis-acting roles in the infection cycles of these viruses[1]. Many internally encoded stem-loop structures were shown to exert diverse functions ranging from templating the synthesis of uridylylated VPg (viral protein genome-linked), anchoring viral RNA-dependent RNA polymerase (RdRP), and serving as the initiation site of genome encapsidation[6,7,8] Despite their recognized roles in viral multiplication cycles, RNA secondary structures have so far been studied in only a relatively small number of model viruses. We show that the stem portion of SL1 extends into the coding region of RNA1 polyprotein, contributing to the previous failure to complement SLC function with solely RNA1 5′ UTR Both SL1 and SLC are required for the accumulation of their respective genome segments in infected cells, likely through their participation in the step of genome replication
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